RS51468B - COMPLETE HUMAN ANTIBODIES FOR HUMANS 4-1BB (CD137) - Google Patents

Abstract

The fully human monoclonal antibody or antigen-binding portion thereof, which specifically binds to human 4-1BB and which contains a light chain variable region and a heavy chain variable region, wherein: said light chain variable region comprises a CDR1 containing amino acid residues 44-54 of SEQ ID NO: 6, CDR2 comprising amino acid residues 70-76 of SEQ ID NO: 6 and CDR3 comprising amino acid residues 109-119 of SEQ ID NO: 6; said heavy chain variable region comprises CDR1 comprising amino acid residues 50-54 of SEQ ID NO: 3, CDR2 comprising amino acid residues 69-84 of SEQ ID NO: 3 and CDR3 comprising amino acid residues 117-129 of SEQ ID NO: 3 The application contains 8 more patent claims.

Description

AREA OF DISCOVERY

The invention relates to fully human antibodies and more specifically to fully human antibodies to human 4-IBB (CD137).

BASIS OF THE INVENTION

A large body of evidence shows unequivocally that there is some degree of immune response to cancer in humans and animals. In cancer patients, cellular components of the immune system are capable of recognizing antigens expressed by tumor cells, such as differentiation of oncofetal antigens or mimic gene products (S. Rosenberg, Nature, 411: 380-4 (2001); P. van der Bruggen et al., Immunological Rev., 188: 51-64 (2002)). A number of clinical trials have shown that tumor-infiltrating lymphocytes have a favorable prognostic significance (E. Halapi, Med. Oncol., 15 (4): 203-11 (1998); Y. Naito et al., Cancer Res., 58 (16): 3491-4 (1998); L. Zhang et al., N. E. J. Med., 348 (3): 203-13 (2003)). In addition, treatment with immunomodulators, such as cytokines or bacterial products, cancer vaccines or adoptive immunotherapy, has led to tumor regression in a number of patients (S. Rosenberg, Cancer J. Sci. Am. 6 (S): 2 (2000); P. Bassi, Surg. Oncol., 11 (1-2): 77-83 (2002); S. Antonia et al., Current Opinion in Immunol., 16: 130-6 (2004)). Despite these responses, cancer-directed immunity often fails to effectively eliminate tumor cells. The causes of this failure can be divided into three main categories: (i) reduced tumor recognition by immune cells, either due to variable expression of tumor antigens or reduced expression of major histocompatibility complex (MHC) class I; (ii) an immunosuppressive tumor microenvironment, as a result of secretion of inhibitory cytokines by tumor cells (eg, TGF-β); and (iii) poor tumor immunogenicity as a consequence of lack of expression of costimulatory molecules on tumor cells, resulting in the inability of tumor cells to efficiently stimulate T-cells. Advances in our understanding of the conditions required for tumor antigen recognition and immune effector functions indicate that a potential strategy for augmenting the anti-tumor immune response is the provision of costimulation via a helper molecule. In order for tumor antigen-specific T-cells to initiate and maintain effector functions, their co-stimulation is required. Therefore, therapies that target costimulatory molecules can be applied to modulate and enhance the immune response to tumors.

In the current model for T-cell activation, it is assumed that two signals are required for full activation of natural T-cells: (i) a signal generated by the binding of processed antigens presented on the T-cell recipe to a major histocompatibility complex (MHC) class I molecule; and (ii) an additional signal resulting from the interaction of costimulatory molecules on the surface of T-cells and their ligands on antigen-presenting cells. Recognition of antigen by natural T-cells is insufficient by itself to trigger T-cell activation. Without a costimulatory signal, T-cells can be eliminated either by death or induction of anergy. Signal transduction via the CD28 costimulatory molecule appears to be critical for the initiation of T-cell responses. However, it has been shown that signal transmission via CD137 (4-IBB) is the basis for the maintenance and expansion of the immune response to antigens, as well as for the generation of memory T-cells.

CD137 (4-IBB) is a member of the tumor necrosis factor receptor (TNF-R) gene family, which includes proteins involved in the regulation of cell proliferation, differentiation and programmed cell death. CD137 is a 30 kDa type I membrane glycoprotein that is expressed as a 55 kDa homodimer. The receptor was first described in mice (B. Kwon et al., P. N. A. S. USA, 86: 1963-7 (1989)) and later identified in humans (M. Alderson et al., Eur. J. Immunol., 24: 2219-27 (1994); Z. Zhou et al., Immunol. Lett., 45: 67 (1995)) (See, also, published PCT applications W095/07984 and W096/29348, and U.S. Patent No. 6,569,997, which are referenced herein (See, SEQ ID NO: 2.)). Human and murine forms of CD137 are 60% identical at the amino acid level. Conserved sequences occur in the cytoplasmic domain as well as 5 other regions of the molecule, suggesting that these molecules may be important for the function of the CD 137 molecule (Z. Zhou et al., Immunol. Lett., 45: 67 (1995)). It has been shown that CD137 expression is predominantly present on cells of lymphoid origin, such as activated T-cells, activated natural killer (NK) cells, NKT-cells, CD4CD25 T-cell regulatomes, and also on activated thymocytes and intraepithelial lymphocytes. In addition, CD137 has also been shown to be expressed on cells of myeloid origin, such as dendritic cells, monocytes, neutrophils, and eosinophils. Even though CD137 expression is mostly restricted to immune/inflammatory cells, there have been papers describing its expression on endothelial cells associated with a small number of tissues from sites of inflammation and tumors.

The functional activity of CD137 on T-cells has been extensively described. Signaling via CD 137 in the presence of suboptimal doses of anti-CD3 has been shown to induce T-cell proliferation and cytokine synthesis (mainly lFN-γ), as well as to inhibit activated cell death. These effects have been reported in both murine and human T-cells (W. Shuford et al., J. Exp. Med., 186 (1) : 47-55 (1997); D. Vinay et al., Semin. Immunol., 10 (6): 481-9 (1998); D. Laderach et al., Int. Immunol., 14 (10): 1155-67. (2002)). In both humans and mice, costimulation enhances effector functions, such as IFN-γ production and cytotoxicity, by increasing the number of antigen-specific and effector CD8+ T-cells. In the absence of anti-CD3 signaling, CD137 stimulation does not alter T-cell function, indicating that CD 137 is a costimulatory molecule.

Physiological events observed after CD 137 stimulation on T-cells are mediated through NF-kB and PI3K/ERK1/2 signals with distinct physiological functions. NF-κB signals drive the expression of BC1-xl, an anti-apoptotic molecule, resulting in increased survival, with PI3K and ERK1/2 signals specifically responsible for CD 13 7-mediated cell cycle progression (H. Lee et al., J. Immunol., 169 (9): 4882-8

(2002)). The effect of CD137 activation on the inhibition of activation-induced cell death was demonstrated in vitro by Hurtado et al. (J. Hurtado et al., J. Immunol., 158 (6): 2600-9 (1997)), and in an in vivo system in which anti-CD137 monoclonal antibodies (mabs) were shown to produce long-term survival of superantigen-activated CD8+ T-cells by preventing clonal deletion (C. Takahashi et al., J. Immunol., 162: 5037 (1999)). Later, two papers showed, under different experimental conditions, that CD137 signaling regulated both the clonal expansion and survival of CD8+ T-cells (D. Cooper et al., Eur. J. Immunol., 32 (2): 521-9

(2002); M. Maus et al., Nat. Biotechnol., 20: 143 (2002)). The reduced apoptosis observed after costimulation was correlated with increased levels of Bc1-xlu CD8+ T-cells, while Bcl-2 expression remained unchanged. The up-regulation of the anti-apoptotic genes Bcl-<XL>and bfl-1 by 4-1BB was shown to be mediated through the activation of NF-kB, since PDTC, an NF-KB-specific blocker, inhibited the 4-lBB-mediated up-regulation of Bc1-Xl (H. Lee et al., J. Immunol., 169 (9): 4882-8 (2002)). On the other hand, clonal expansion of activated T-cells appears to be mediated by increased expression of cyclins D2, D3 and E, as well as down-regulation of p27<k>,<p1> proteins. This effect occurs in both an IL-2-dependent and -independent manner (H. Lee et al., J. Immunol., 169 (9): 4882-8 (2002)).

Collectively, CD 137 stimulation results in increased expansion, survival, and effector functions of newly generated CD8+ T-cells, acting, in part, directly on these cells. CD4+ and CD8+ T-cells have been shown to respond to CD137 stimulation, however. the increase in T-cell function appears to be greater in CD8+ cells (W. Shuford et al., J. Exp. Med., 186 (1): 47-55 (1997); I. Gramaglia et al., Eur. J. Immunol., 30 (2): 392-402 (2000); C. Takahashi et al., J. Immunol., 162: 5037 (1999)). Based on the crucial role of CD137 stimulation in the function and survival of CD8+ T-cells, manipulation of the CD137/CD137L system provides a possible approach for the treatment of tumors and viral pathogen infections.

Constitutive expression of CD 137 on freshly isolated dendritic cells (DCs) was recently demonstrated in mice (R. Wilcox et al., J. Immunol., 169 (8): 4230-6

(2002); T. Futagava et al., Int. Immunol., 14 (3): 275-86 (2002)) and humans (S. Pauly et al., J. Leukoc. Biol. 72 (1) : 35-42 (2002)). These works showed that stimulation of CD137 on DCs resulted in secretion of IL-6 and IL-12, and more importantly, increased the ability of DCs to stimulate T-cell responses to alloantigens. In addition, Pan et al. demonstrated that CD 137 signaling in DCs resulted in upregulation of MHC Class I and costimulatory molecules, and produced an increased ability of DCs to infiltrate tumors (P. Pan et al., J. Immunol., 172 (8): 4779-89 (2004)). Thus, costimulation of CD137 on DCs appears to provide a novel pathway for DC proliferation, maturation, and migration.

Activated natural killer (NK) cells express CD 137 after cytokine stimulation (I. Melero et al., Cell Immunol., 190 (2): 167-72 (1998); R. Wilcox et al., J. Immunol., 169 (8): 4230-6 (2002)). Several papers have shown that NK cells appear to be crucial for the modulation of the antitumor immune response induced by agonistic CD137 antibodies ((I. Melero et al., Cell Immunol., 190 (2): 167-72 (1998); R. Miller et al., J. Immunol., 169 (4): 1792-800 (2002); R. Wilcox et al., J. Immunol., 169 (8): 4230-6 (2002)).The elimination of NK cells significantly reduces the antitumor activity of NK cells. Binding of CD137 on NK cells induces proliferation and secretion of IFN-γ. Namely, in vitro, CD137-stimulated NK cells display immunoregulatory or "helper" activity for CD8+ cytolytic T cells. T-cell Therefore, signal transmission via CD137 on NK cells can modulate innate immunity to tumors.

A paradoxical effect has been described for CD 137 stimulation in that agonistic CD 137 antibodies can induce suppression of humoral responses to T-cell antigens in primate and mouse models (H. Hong et al., J. Immunother., 23 (6): 613-21 (2000); R. Mittler et al., J. Exp. Med., 190 (10): 1535-40 (1999)). Specifically, CD137 agonistic antibodies have been shown to produce significant relief of symptoms associated with antibody-dependent autoimmune diseases such as systemic lupus erythematosus and experimental autoimmune encephalomyelitis (J. Foell et. al., N. Y. Acad. Sci., 987: 230-5

(2003); Y. Sun et al., Nat. Med., 8 (12): 1405-13 (2002)). Recently, Seo et al. have shown, in a mouse model of rheumatoid arthritis, that treatment with an agonistic anti-CD137 antibody prevented the development of the disease, as well as significantly halted the progression of the disease (S. K. Seo et al., Nat. Med. 10; 1099-94 (2004)). The mechanism responsible for this effect is not well defined, but in a model of rheumatoid arthritis it was shown that treatment with a CD137 agonistic antibody resulted in the expansion of IFN-γ-producing CD11C-CD8+ T cells. IFN-γ then stimulates dendritic cells to produce indoleamine-2,3-dioxygenase (IDO), which has immunosuppressive activity. It has also been hypothesized that CD 137 signaling on antigen-activated CD4+ T-cells results in the induction of IFN-γ secretion that activates macrophages. Activated macrophages then produce death signals for B cells. Continuous signaling through CD 137 on CD4+ T-cells can then induce activation-induced cell death (AICD) of these CD4+ activated T-cells. Thus, by eliminating antigen-activated T-cells and B-cells, a reduced antibody response was noted and, as a consequence, a remarkable reduction in Th2-mediated inflammatory diseases was noted (B. Kwon et al., J. Immunol., 168 (11): 5483-90 (2002)). These studies suggest a role for the administration of agonistic CD 137 antibodies in the treatment of inflammatory or autoimmune diseases, without inducing general suppression of the immune system.

The natural ligand for CD137, CD137 ligand (CD137L), a 34kDa glycoprotein member of the TNF superfamily, is mainly detected on activated antigen-presenting cells (APCs), such as B cells, macrophages, dendritic cells and also on murine B-cell lymphomas, activated T-cells, as well as human carcinoma lines of epithelial origin (R. Goodwin et al., Eur. J. Immunol., 23 (10): 2631-41 (1993); Z. Zhou et al., Immunol. Lett., 45: 67

(1995); H. Salih et al., J. Immunol., 165 (5): 2903-10 (2000)). Human CD137L shares 36% homology with its murine counterpart (M. Alderson et al., Eur. J. Immunol., 24: 2219-27

(1994)).

In addition to signaling to CD137-expressing cells, binding of CD137 to CD137L initiates a bidirectional signal that results in functional effects on CD137L-expressing cells. Langstein et al. showed that the binding of CD137-Ig fusion protein to CD137L on activated monocytes induces the production of IL-6, IL-8 and TNF-a, correspondingly regulates ICAM and inhibits IL-10, which results in increased adherence (J. Langstein et al.,

.1. Immunol., 160 (5): 2488-94 (1998)). Besides that. monocyte proliferation was noted along with a higher rate of apoptosis (J. Langstein et al., J. Leukoc. Biol., 65 (6): 829-33

(1999)). These observations were confirmed by the tests performed by Ju et al. (S. Ju et al., Hybrid Hybridomics, 22(5):333-8 (2003)), in which a functional anti-CD137L antibody was shown to induce a high proliferation rate of peripheral blood monocytes. Blocking the ligand resulted in inhibition of T-cell activation. In addition, soluble CD137L has been found in the serum of patients with rheumatoid arthritis and hematological malignancies (H. Salih et al., J. Immunol., 167 (7): 4059-66 (2001)). Thus, the interaction of CD137 with CD137L produces and influences functional effects on T-cells and APCs.

In another important aspect of T-cell function, it was shown that agonistic anti-CD137 antibodies preserved T-cell responses to protein antigens in aged mice. An age-related decline in the immune response to antigens, a process known as immunosenescence, is well established (R. Miller, Science, 273: 70-4 (1996); R. Miller, Vaccine, 18: 1654-60 (2000); F. Hakim et al., Curr. Opinion Immunol., 16: 151-156

(2004)). This phenomenon appears to be due to a change in the balance between the magnitude of cell expansion and cell survival or death. Bansal-Pakala et al. have tested the hypothesis that secondary costimulation via CD137 can be used to enhance T-cell responses in situations where T-cells do not receive sufficient stimulation, as a result of either reduced expression of CD3 or CD28, or reduced signal quality. Their studies showed that aged mice had an incomplete in vitro response compared to young mice (P. Bansal-Pakala et al., J. Immunol., 169 (9): 5005-9 (2002)). However, when aged mice were treated with anti-CD137 monoclonal antibodies, T-cell proliferative and cytokine responses were identical to those seen in young mice. Although the specific mechanism responsible for this effect has not been elucidated, it has been speculated that increased expression of anti-apoptotic molecules similar to Bc1-xli stimulation of IL-2 secretion in vivo may play a role in restoring abnormal T-cell responses. These studies demonstrated the potential of agonistic anti-CD137 antibodies to restore weak T-cell responses in elderly immunocompromised individuals, which has great implications for the use of anti-CD137 antibodies in cancer patients.

The role of CD137 targeted therapy in cancer treatment has been suggested in in vivo efficacy studies in mice using agonistic anti-mouse CD137 monoclonal antibodies. In a paper by Melero et al., an agonistic anti-mouse CD137 antibody produced a cure in a P815 mast cell tumor as well as in a low-immunogenic Agi04 tumor model (I. Melero et al., Nat. Med., 3(6):682-5 (1997)). Both CD4+ and CD8+ T-cells, as well as NK cells, are required for anti-tumor activity, given that selective in vivo elimination of each subpopulation resulted in a reduction or complete loss of anti-tumor activity. It was also shown that minimal induction of an immune response was necessary for anti-CD137 therapy to be effective. Several investigators have used anti-CD137 antibodies to demonstrate the feasibility of this approach for cancer treatment (J. Kim et al., Cancer Res., 61 (5): 2031-7 (2001); O. Martinet et al., Gene Ther., 9 (12): 786-92 (2002); R. Miller et al., J. Immunol., 169 (4): 1792-800 (2002); R. Wilcox et al., Cancer Res., 62 (15): 4413-8 (2002)).

In support of the results of anti-tumor efficacy of administration of agonistic CD 137 antibodies, signals originating from CD137L have been shown to induce CTL activity and anti-tumor responses (M. DeBenedette et al., J. Immunol., 163 (9): 4833-41 (1999); B. Guinn et al., J. Immunol., 162 (8): 5003-10 (1999)). Several papers have shown that gene transfer of CD137 Uganda into murine carcinomas resulted in tumor reduction, indicating the need for costimulation in the generation of an effective immune response (S. Mogi et al., Irnmunologv, 101 (4): 541-7 (2000); I. Melero et al., Cell Immunol., 190 (2): 167-72 (1998); B. Guinn et al., J. Immunol., 162(8):5003-10 (1999)). Salih et al. reported the expression of CD137L in human carcinomas and human carcinoma cell lines (H. Salih et al., J. Immunol., 165(5):2903-10 (2000)) and showed that tumor cells expressing the ligand were able to transmit a costimulatory signal to T-cells, resulting in the release of IFN-y and IL-2, and that this effect was correlated with the levels CD137L on tumors. It is not known whether expression of CD137L in human tumors may render these tumors more sensitive to agonistic CD 137 antibodies.

In CD137L-/- mice, the importance of the CD137/CD137L system in T-cell responses to both viruses and tumors was emphasized (M. DeBenedette et al., J. Immunol., 163 (9): 4833-41

(1999); J. Tan et al., J. Immunol., 164(5): 2320-5 (2000); Kwon et al., J. Immunol., 168(11):5483-90 (2002)). Studies using CD137 and CD137L knockout mice have demonstrated the importance of CD 137 costimulation in graft-versus-host disease as well as anti-viral cytolytic T-cell responses. Mice lacking the CD 137 gene had increased T-cell proliferation but also decreased cytokine production and cytotoxic T-cell activity (B. Kwon et al., J. Immunol., 168 (11): 5483-90 (2002); D. Vinay et al., Immunol. Cell Biol., 81 (3): 176-84 (2003)). More recently, it was shown that knockout mice (CD137-/-) had a higher frequency of tumor metastases (4-fold) compared to control mice. These results suggest that restoration of CD137 signaling using agonistic anti-CD137 antibodies is a suitable approach to enhance cellular immune responses to viral pathogens and cancers.

In addition to results in in vivo mouse models supporting the involvement of CD 137 signaling in antitumor immune responses, studies performed in primary human tumor samples confirmed the role of CD137 in the production of effector T-cells. In patients with Ewing sarcoma, Zhang et al. have shown that intratumoral effector T-cells present a CD3+/CD8+/CD28-/CD137+ phenotype. Unexpectedly, the simultaneous existence of progressive tumor growth and anti-tumor immunity (effector T-cells) was noted. Ex vivo stimulation studies with patient cells showed that tumor-induced T-cell proliferation and activation required costimulation with CD137L. Stimulation of PBL with anti-CD3/CD137L, but not anti-CD3/anti-CD28, induced tumor lytic effectors. These studies provided further evidence that CD 137-mediated costimulation can result in the expansion of tumor-reactive CTLs (H. Zhang et al, Cancer Biol. Ther., 2(5):579-86 (2003)). In addition, CD137 expression has been demonstrated in tumor-infiltrating lymphocytes in hepatocellular carcinomas (HCC) (Y. Wan et al., World J. Gastroenterol, 10(2):195-9 (2004)). CD 137 expression was determined in 19 out of a total of 19 HCCs using RT-PCR, as well as in 13/19 using immunofluorescent staining. Conversely, CD137 was not found in peripheral mononuclear cells from the same patients. Analyzes performed with liver tissues from healthy donors failed to demonstrate CD137 expression. These trials did not attempt to correlate clinical disease with CD137 expression. Thus, studies performed on Ewing sarcoma and hepatocellular carcinoma revealed the presence of CD137-expressing TILs, with concomitant disease progression. In Ewing sarcoma, it was shown that CD137+TILs were capable of killing tumor cells ex vivo, indicating that the CD 137 pathway was intact in these patients and that suppressive factors in the tumor microenvironment may have inhibited their function. Hence, it can be hypothesized that systemic administration of agonistic CD 137 antibodies may provide the signal necessary for the expansion of these effector T-cells.

In addition to their role in the development of immunity to cancer, experimental results support the use of CD 137 agonist antibodies for the treatment of autoimmune and viral diseases (B. Kvvon et al, Exp. Mol. Med., 35 (1): 8-16 (2003); H. Salih et al, J. Immunol., 167 (7): 4059-66 (2001); E. Kvvon et al., P. N. A. S. USA, 96: 15074 (1999); J. Acad. Sci., 8 (12): 1099-94.

Thus, based on the role that 4-IBB plays in modulating the immune response, it would be desirable to produce anti-human 4-IBB antibodies with agonistic activities that can be used to treat or prevent human diseases such as cancer, infectious diseases, and autoimmune diseases.

BRIEF SUMMARY OF THE INVENTION

The present invention provides fully human antibodies that bind to human 4-1BB (H4-1BB) and that enable binding of H4-1BB to human 4-IBB ligand (H4-1BBL). Therefore, the invention relates to antibodies that bind to H4-1BB and that do not block the binding of H4-1BB to H4-1BBL, thereby enabling the binding of both the antibody of the invention and H4-1BBL to H4-1BB. The invention also provides antibodies with agonistic effects in that antibody binding to H4-1BB results in enhancement and stimulation of H4-1BB mediated immune responses. These antibodies can be used as immuno-enhancers of the anti-tumor or anti-viral immune response, or as immunomodulators of autoimmune diseases that are mediated by T cells. These antibodies can also be used as diagnostic tools to detect H4-1BB in the blood or tissues of patients with cancer, autoimmune or other diseases.

In one aspect, the invention provides a monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to H4-1BB and that comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3), as shown in FIG. 4, and the heavy chain variable region comprises CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3), as shown in FIG. 3 or FIG. 7. The monoclonal antibody (mab) can be, for example, an IgG4 antibody or an IgG1 antibody.

In another aspect, the invention provides a monoclonal antibody, or antigen-binding portion thereof, wherein the light chain comprises a variable region as shown in FIG. 4, and the heavy chain comprises a variable region as shown in FIG. 3 or FIG. 7.

In another aspect, the invention provides a monoclonal antibody comprising a light chain and a heavy chain, wherein the light chain comprises amino acid residues 21-236 of SEQ ID NO: 6 and the heavy chain comprises amino acid residues 20-467 of SEQ ID NO: 3. In another aspect, the invention provides a monoclonal antibody comprising a light chain and a heavy chain, wherein the light chain comprises amino acid residues 21-236 of SEQ ID NO: 3 ID NO: 6 and the heavy chain comprises amino acid residues 20-470 of SEQ ID NO: 9.

Antibodies according to the invention have a wide therapeutic application as immunomodulators of diseases such as cancer, autoimmune diseases, inflammatory diseases and infectious diseases.

The invention further provides methods for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an antibody of the invention. In one embodiment, the method further comprises administering a vaccine. Suitable vaccines include, for example, a tumor cell vaccine, a DNA vaccine, a GM-CSF-modified tumor cell vaccine, or an antigen dendritic cell vaccine. The cancer can be, for example, prostate cancer, melanoma or epithelial cancer.

In a further aspect, the invention provides a method for enhancing an immune response, comprising administering an antibody according to the invention and a SIV gag vaccine. In a further aspect, the invention provides a method for enhancing an immune response, comprising the administration of an antibody according to the invention and a PSA vaccine. In a further aspect, the invention provides a method for enhancing an immune response to an SIV gag vaccine, comprising administering an antibody of the invention. In a further aspect, the invention provides a method for enhancing an immune response to a PSA vaccine, comprising administering an antibody according to the invention.

The invention also provides pharmaceutical compositions comprising an antibody according to the invention or an antigen-binding portion thereof, as well as a pharmaceutically acceptable carrier. The pharmaceutical composition may be administered alone or in combination with an agent, eg, a cancer treatment agent such as a chemotherapeutic agent, a vaccine, or another immunomodulatory agent.

The invention also provides isolated polynucleotides comprising a nucleotide sequence selected from: (a) nucleotides encoding the amino acid sequence of amino acid residues 20-467 of SEQ ID NO: 3; (b) nucleotides encoding the amino acid sequence SEQ ID NO: 3; (c) nucleotides encoding the amino acid sequence of amino acid residues 21-236 of the sequence SEQ ID NO: 6; (d) nucleotides encoding the amino acid sequence SEQ ID NO: 6; (e) nucleotides encoding the amino acid sequence of amino acid residues 20-470 of SEQ ID NO: 9; (f) nucleotides encoding the amino acid sequence SEQ ID NO: 9; and (g) nucleotides encoding a fragment of the amino acid sequence from (a) to (f), such as a variable region, a constant region, or one or more CDRs. The isolated polynucleotides according to the invention further comprise nucleotide sequences encoding at least one CDR with SL. 3, at least one CDR of FIG. 4 or at least one CDR of FIG. 7. The invention further provides isolated polynucleotides comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 7.

The invention also provides polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9. In a further aspect, the invention provides isolated polypeptides comprising the amino acid sequence of amino acid residues 20-467 of SEQ ID NO: 3, isolated polypeptides comprising the amino acid sequence of amino acid residues 21-236 of SEQ ID NO: 6 and isolated polypeptides comprising the amino acid sequence of aminoxyl residues 20-470 of SEQ ID NO: 9. In a further aspect, the invention provides isolated polypeptides comprising the amino acid sequence of at least one CDR with SL. 3, FIG. 4 or FIG. 7, or at least the variable or constant region of FIG. 3, FIG. 4 or FIG. 7.

The invention further includes an immunoglobulin that specifically binds to H4-1BB, wherein said immunoglobulin comprises an antigen-binding region. In one embodiment, the immunoglobulin is a Fab or F(ab')2 fragment of an antibody of the invention.

The invention also includes a cell line that produces an antibody or an antigen-binding portion thereof according to the invention, recombinant expression vectors comprising nucleotides according to the invention, and methods for producing an antibody according to the invention by culturing an antibody-producing cell line.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a map of plasmid pD17-20H4.9.h4a.

FIG. 2 shows a map of plasmid pD16gate-20H4.9.LC.

FIG. 3 (FIG. 3A-3H) shows the nucleotide sequence of plasmid pD17-20H4.9.h4a, including the coding strand (SEQ ID NO: 1), the complementary strand (SEQ ID NO: 2) and the amino acid sequence (the leader peptide is represented by amino acid residues 1-19 of SEQ ID NO: 3; the heavy chain is represented by amino acid residues 20-467 of SEQ ID NO: 3) which encodes the encoding chain.

FIG. 4 (FIGS. 4A-4F) shows the nucleotide sequence of plasmid pD16gate-20H4.9.LC, including the coding strand (SEQ ID NO: 4), the complementary strand (SEQ ID NO: 5) and the amino acid sequence (the leader peptide is represented by amino acid residues 1-20 of SEQ ID NO: 6; the light chain is represented by amino acid residues 21-236 of SEQ ID NO: 6) which encodes the encoding chain.

FIG. 5 is a schematic representation of the 20H4.9-IgG1 heavy chain sequence construct.

FIG. 6 is a schematic representation of the 20H4.9 light chain sequence construct.

FIG. 7 (FIGS. 7A-7D) shows the nucleotide and amino acid sequences of the 20H4.9-IgGl heavy chain construct, including the coding strand (SEQ ID NO: 7), the complementary strand (SEQ ID NO: 8) and the aminoselin sequence (the leader peptide is represented by amino acid residues 1-19 of SEQ ID NO: 9; the heavy chain is represented by amino acid residues 20-470 of SEQ ID NO: 9) encoded by the encoding chain.

FIG. 8 (FIG. 8A-8B) illustrates the results obtained by the binding of monoclonal antibody 20H4.9-IgGl to human CD137 using ELISA (FIG. 8A) and the effect of monoclonal antibody 20H4.9-IgGl on the interaction of CD137-CD137L (FIG. 8B).

FIG. 9 (FIGS. 9A-9B) illustrates the results obtained by binding of the monoclonal antibody 20H4.9-IgG1 to human or macaque monkey cells stimulated with PMA-ionomycin. Human CEMs (FIG. 9A) or monkey PBMCs (FIG. 9B) were incubated with 20H4.9-IgG1 or human CD137L fusion protein.

FIG. 10 (FIGS. 10A-10B) illustrates the results obtained from the induction of IFN-γ in costimulatory assays with anti-CD137 antibodies, which are expressed as the number of fold increases over controls in pg/ml. Due to variable baseline response among donors, results were normalized to control treatments (=1). The mean baseline IFN-γ level for human T-cells (FIG. 10A) or monkey PBMCs (FIG. 10B) stimulated with anti-CD3 alone was 592 pg/ml and 505 pg/ml, respectively.

FIG. 11 shows the plasmon resonance diagram of the binding of monoclonal antibody 20H4.9-IgG4 and monoclonal antibody 20H4.9-IgG1 to human CD137.

FIG. 12 illustrates concentration-dependent binding of 20H4.9-IgG4 to human CEM cells stimulated with PMA ionomycin, but not binding to unstimulated CEM cells.

FIG. 13 (FIG. 13A-B) illustrates the induction of IFN-γ in costimulatory assays with anti-CD137 antibodies. The results are expressed as the number of times the values increased compared to the controls in pg/ml. Due to variable baseline response among donors, results were normalized to control treatments (=1). The mean baseline IFN-γ level for human T-cells (FIG. 13A) or monkey PBMCs (FIG. 13B) stimulated with anti-CD3 alone was 592 pg/ml and 505 pg/ml, respectively.

FIG. 14 (FIG. 14A-14B) illustrates the results obtained from the dose-dependent increase in IFN-γ synthesis under the influence of monoclonal antibodies 20H4.9-IgG4 (FIG. 14A) and the effect of antibody cross-linking by the addition of cross-linking anti-human IgG antibody (7 ug/ml) (FIG. 14B).

FIG. 15 illustrates the effect of monoclonal antibody 20H4.9-IgG4 on T-cell survival and cell cycle progression. Human T-cells were co-stimulated with anti-CD3 (1 µg/ml) ± monoclonal antibody 20H4.9-IgG4 at the indicated concentrations. Six days after the start of the assay, cells were harvested and stained with Annexin-V and propidium iodide to determine the number of viable cells (Annexin V/PI negative) or PE-conjugated cyclin D2 to determine cells in the cell cycle. Results represent the mean (SD) of 4 groups of 20H4.9-IgG4 monoclonal antibodies tested in parallel.

FIG. 16 (FIGS. 16A-16D) shows the antigen-specific IFN-γ response in macaque monkeys as measured by ELISPOT after treatment with DNA vaccine ± anti-human 4-1BB antibodies. Animals were treated with SIV gag vaccine (day 0, 28, 56; FIG. 16A), SIV gag vaccine (day 0, 28, 56) and monoclonal antibody 20H4.9-IgG4 (day 12, 15, and 19; FIG. 16B), or SIV gag vaccine (day 0, 28, 56) and hu39E3.G4 (day 12, 15 and 19; SL 16C). One group of animals was not treated (FIG. 16D). At various times after treatment, blood was drawn and PBMCs were isolated, then their ability to secrete IFN-γ in the presence of antigenic stimulation was determined.

DETAILED DESCRIPTION OF THE PROGRESS

The invention relates to the preparation and characterization of antibodies and antigen-binding fragments thereof (including fusion proteins containing an antigen-binding fragment of an antibody according to the invention), for use in the treatment of diseases, such as cancer, infectious disease, inflammatory disease or autoimmune disease. The cancer can be, for example, prostate cancer, melanoma or epithelial cancer.

Antibodies are capable of binding to H4-1BB and can have high affinity for H4-1BB and effectively enhance T cell responses. In one embodiment, the antibody induces IFN-γ production in costimulatory assays, but does not affect the binding of H4-1BB to its corresponding ligand, H4-1BBL, and does not bind complement.

Antibodies of the invention can be produced by methods well known in the art. In one embodiment, antibodies can be produced by expression in transfected cells, such as immortal eukaryotic cells, such as myeloma or hybridoma cells.

Antibodies of the invention may be used alone, or in combination with other therapeutic agents such as radiotherapy (including radiation), hormonal therapy, cytotoxic agents, vaccines and other immunomodulatory agents, such as cytokines and biological response modifiers. These agents are particularly useful for the treatment of cancer and immuno-proliferative disorders.

In one embodiment, the invention provides a monoclonal antibody (mab) 20H4.9-IgG4. FIG. 1 and 2 provide maps of plasmids pD17-20H4.9.h4a and pD16gate-20H4.9.LC, respectively, which can be used to produce monoclonal antibody 20H4.9-IgG4. FIG. 3 (FIGS. 3A-3H) provides the nucleotide sequence of plasmid pD17-20H4.9.h4a, including the coding strand (SEQ ID NO: 1), the complementary strand (SEQ ID NO: 2) and the amino acid sequence (the leader peptide is represented by amino acid residues 1-19 of SEQ ID NO: 3; the heavy chain is represented by amino acid residues 20-467 of SEQ ID NO: 3) which encodes the encoding chain. FIG. 4 (FIGS. 4A-4F) shows the nucleotide sequence of plasmid pD16gate-20H4.9.LC, including the coding strand (SEQ ID NO: 4), the complementary strand (SEQ ID NO: 5) and the amino acid sequence (the leader peptide is represented by amino acid residues 1-20 of SEQ ID NO: 6; the light chain is represented by amino acid residues 21-236 of SEQ ID NO: 6) which encodes the encoding chain.

In another aspect, the invention provides a monoclonal antibody (mab) 20H4.9-IgG1. FIG. 5 provides a schematic representation of the heavy chain sequence construct of the monoclonal antibody 20H4.9-IgG1. FIG. 6 is a schematic representation of the light chain sequence construct of monoclonal antibody 20H4.9, for monoclonal antibody 20H4.9-IgG4 and 20H4.9-IgG1. FIG. 7 provides the nucleotide sequence (coding strand (SEQ ID NO: 7) and complementary strand (SEQ ID NO: 8)) of the heavy chain sequence construct of FIG. 5, as well as the amino acid sequence (the leader peptide is represented by amino acid residues 1-19 of SEQ ID NO: 9; the heavy chain is represented by amino acid residues 20-470 of SEQ ID NO: 9) encoded by the coding chain. The light chain of monoclonal antibody 20H4.9-IgG1 is the same as the light chain of monoclonal antibody 20H4.9-IgG4.

The invention also encompasses antibodies with conservative amino acid substitutions from the heavy and light chain amino acid sequences shown in SEQ ID NOS: 3, 6 and 9, which generally have no effect on H4-1BB binding. Conservative substitutions usually involve the substitution of one amino acid for another with similar properties, eg, substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

Polynucleotides encoding the polypeptides of the invention typically additionally contain an expression control sequence operably linked to the polypeptide encoding sequences, including native or heterologous promoter regions. Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts can also be used. Once the vector has been incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequences and for, if desired, collection and purification of light chain, heavy chain, light/heavy chain dimer, or intact antibody, which may be followed by binding of fragments or other immunoglobulin forms. (See, S. Bevchok, Cells of Immunoglobulin Synthesis, Academic Press, N.Y. (1979)). Single chain antibodies or minibodies (single chain antibodies fused to one or more CH domains) can also be produced by fusing the nucleic acid sequences encoding the VL and VH regions described herein to DNA encoding the polypeptide linker.

Prokaryotic hosts, such as E. coli, and other microbes, such as yeast, can be used to express the antibodies of the invention. In addition to microorganisms, mammalian tissue cell cultures can also be used for the expression and production of antibodies according to the invention. Eukaryotic cells may be preferred due to the number of suitable host cell lines developed that are capable of secreting intact immunoglobulins including, for example, CHO (Chinese hamster ovary) cell lines, COS (African green monkey fibroblast cell line), HeLa cells, myeloma and hybridoma cell lines. Expression vectors for these cells may include expression control sequences, such as a promoter or enhancer, as well as necessary informational processing sites, such as ribosome binding sites, RNA cleavage sites, polyadenylation sites, and transcription termination signaling sequences, as is well known in the art.

Vectors containing the DNA segments of interest (eg, heavy and/or light chain coding sequences and expression control sequences) can be transferred into a host cell using well-known procedures, which vary depending on the type of host cell. For example, calcium chloride transfection is often used for prokaryotic cells, while calcium phosphate treatment, lipofection, or electroporation can be used for other cellular hosts. (See, e.g., T. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1982)).

Once expressed, antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms, can be purified by standard procedures in the art, such as ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. Generally pure immunoglobulins of at least 90 to 95% homogeneity are preferred, with homogeneity of 98 to 99% or greater being more preferred.

Antibodies of the invention are useful for modulating immune responses mediated by T cells and antibodies. Typical disease states suitable for treatment include cancers, infectious diseases, inflammatory diseases and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus and myasthenia gravis.

The invention also provides pharmaceutical compositions comprising at least one antibody according to the invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be sterilized by conventional well-known sterilization techniques. Pharmaceutical compositions may also contain pharmaceutically acceptable excipients as needed for physiological-like conditions, such as pH-adjusting and buffering agents, stability-enhancing agents such as mannitol or tween 80, toxicity-adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, or human albumin.

The antibodies and pharmaceutical compositions of the invention are particularly useful for parenteral administration, including subcutaneous, intramuscular and intravenous administration. Pharmaceutical compositions for parenteral administration may contain a solution of the antibody dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, as is well known in the art, eg, water, buffered water, saline, glycine, and the like. These solutions are sterile and generally free of particulate material. The formulation of the parenteral composition in the form of a unit dose is particularly useful for ease of administration and uniformity of the dose.

The pharmaceutical composition may additionally contain an additional agent for the treatment of the disease. In one embodiment, the pharmaceutical composition comprises an agent for the treatment of cancer, infectious disease, inflammatory disease, or autoimmune disease. The antibody according to the invention can also be administered simultaneously or separately with an additional agent for the treatment of the disease.

Antibodies of the invention can be used with other means to enhance the immune response to cancer cells in a patient. In one embodiment, the antibody is used in combination with an immunogenic agent, such as cancer cells, purified tumor antigens (including recombinant protein, peptide and carbohydrate molecules), or cells transfected with genes encoding immune-stimulatory cytokines and cell surface antigens. In a further aspect, the antibody is used in combination with a vaccine such as, for example, a tumor cell vaccine, a DNA vaccine, a gene-modified tumor cell vaccine, such as a GM-CSF-modified tumor cell vaccine, a peptide vaccine, or a dendritic cell vaccine with antigens.

Many experimental strategies for tumor vaccination have been found. In one of these strategies, a vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when tumor cells are induced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al., P. N. A. S., 90: 3539-43 (1993); E. Jafee et al., J. Clin. Oncol., 19: 145-56 (2001); R. Salgia et al., J. Clin. Oncol., 21: 624-30 (2003)).

Examination of gene expression and gene expression patterns on a large scale in various tumors has led to the determination of so-called specific tumor antigens (S. Rosenberg, Immunitv 10: 281-7 (1999)). In many cases, these specific tumor antigens are different antigens expressed in tumors and in the cell from which the tumor originated, for example melanocyte antigens gp 100, MAGE antigens, Trp-2. Many of these antigens can be shown to be targeted by tumor-specific T cells found in the host. Antibodies of the invention can be used together with a group of recombinant proteins and/or peptides that are expressed in the tumor to enhance and direct the immune response to these antigens towards a Th1 response. These proteins are normally recognized by the immune system as auto-antigens and therefore have tolerance towards them.

In one aspect of the invention, the antibody is combined with an immunomodulatory agent containing an SIV gag antigen (as a model for an HTV DNA vaccine) or a prostate-specific antigen (PSA), or a DNA vaccine containing a nucleotide sequence encoding a SIV gag antigen or a prostate-specific antigen (PSA). PSA vaccines are described in, for example, M. Pavlenko et al., Br. J. Cancer, 91 (4): 688-94 (2004); J. Wolchok et al., Semin. Oncol., 30 (5): 659-66 (2003); J. Kim et al., Clin. Cancer Res., 7 (3 Suppl.): 882s-889s (2001). SIV gag vaccines are described in, for example, B. Makitalo et al., J. Gen. Viral, 85 (Pt 8): 2407-19 (2004); N. Letvin et al., J. Virol., 78 (14): 7490-7 (2004); S. Mossman et al., AIDS Res. Hum. Retroviruses., 20 (4): 425-34 (2004); F. Bertley et al., J. Immunol., 172(6):3745-57 (2004); L. Patterson et al., J. Virol., 78(5):2212-21 (2004); E. O'Neill et al., AIDS Res. Hum. Retroviruses, 19 (10): 883-90 (2003); Z. Bu et al., Virology, 309 (2): 272-81 (2003).

The tumor antigen may also include, for example, the protein telomerase, which is required for chromosomal telomere synthesis and is expressed in more than 85% of human cancers and only in a limited number of somatic tissues (N. Kim et al., Science, 266, 2011-2013

(1994)). A tumor antigen may also be a "neo-antigen" expressed in cancer cells due to somatic mutations that change the protein sequence or create fusion proteins between two unrelated sequences, or an idiotype from a B cell tumor. Other tumor vaccines may include proteins from viruses implicated in human cancers such as human papillomaviruses (HPV), hepatitis viruses (HBV and HCV), and Kaposi's sarcoma herpesvirus (KHSV). Another form of tumor-specific antigen that can be used with the antibody of the invention is purified heat shock proteins (HSPs) that are isolated from the tumor tissue itself. These heat shock proteins contain protein fragments from tumor cells and these HSPs are highly effective for application to antigen-presenting cells to induce tumor immunity (R. Suot et al., Science 269: 1585-1588 (1995); Y. Tamura et al., Science 278: 117-120(1997)).

Antibodies of the invention can also be used to enhance the immune response to vaccines for viral antigens, such as HIV or HCV. Antibodies of the invention can also be used to enhance the immune response to other immunomodulatory agents, as well as to enhance the memory immune response. Examples of these agents are cytokines such as GM-CSF, IL-2, IL-15, IL-12, F13 ligand, CD40 ligand, adjuvants such as CpG-oligodeoxynucleotide (bacterial DNA) or antibodies to OX-40 or CTLA-4.

Pharmaceutical compositions according to the invention can be applied for prophylactic and/or therapeutic treatments. In therapeutic application, the pharmaceutical composition is applied to a patient who is already ill, in an amount sufficient to cure or at least partially stop the disease. The amount sufficient to achieve this is defined as a "therapeutically effective dose". Amounts effective for this use will depend on the severity of the disease condition and the patient (including, for example, the general condition of the patient's immune system) and may be determined by the skilled artisan. In prophylactic applications, the pharmaceutical composition is administered to a patient who does not have the disease, in order to increase the patient's resistance to the disease. Such an amount is determined as a "prophylactically effective dose". In this application, the exact amounts will depend on the state of health of the patient (including, for example, the general state of the patient's immune system) and can be determined by the skilled practitioner. In one aspect, prophylactic administration is intended to prevent tumor recurrence.

Examples:

Example 1: Antibody production

Material and methods

Fully human monoclonal antibodies to the human CD 137 (4-IBB) receptor were raised in HuMAb-Mouse® mice (Medarex, Inc., Princeton, New Jersey). HuMAb mice were immunized five times intraperitoneally (i.p.) and subcutaneously (s.c.) with 25 µg of the extracellular domain of human CD137 in RIBI adjuvant (Ribi Immunochemical). Before fusion, mice received the same amount of antigen intravenously (i.v.). Spleen cells from immunized mice with appropriate antibody titers to huCD137 were fused to murine myeloma cells according to standard procedure.

Anti-human CD3 monoclonal antibody (clone: HIT3a), human IFN-γ and monkey IFN-γ ELISA kits, cytometric bead array (CBA) kits, and all conjugated antibodies for flow cytometry were obtained from BD Pharmingen (San Diego, California). Human IgGiX and human IgGi were purchased from Sigma-Aldrich (St. Louis, Missouri). CEM cells (ATCC-CRL 2265) were obtained from ATCC. Culture medium (RPMI) and fetal bovine serum (FBS) were purchased from Mediatech Inc. (Herndon, Virginia). Alsever sheep red blood cells were obtained from Colorado Serum Co. (Denver, Colorado).

Selection of hybridomas: Detection of binding to huCD137 by ELISA: To determine hybridomas secreting anti-human CD 137 antibodies, ELISA plates (Nunc MaxiSorp) were coated with human CD137-mouse IgG2b fusion protein at 1<p.>g/ml in PBS overnight at 4°C. Plates were then washed 3 times with PBS with 0.01% Tween-80 (PBS-T) and then blocked with PBS-T plus 1% bovine serum albumin (BSA) for 20 minutes at room temperature. Fifty microliters of supernatants diluted 1:3 in PBS-T were added to the plates and the plates were incubated for 1-2 hours at room temperature. Plates were then washed as before, and antibody binding was detected by incubation with alkaline phosphatase-conjugated goat F(ab')2 anti-human IgG antibody (Jackson Laboratories, West Grove, Pennsylvania). Plates were developed with pNPP and read at 405 nm.

Blocking assay: The ability of twenty-six hybridomas secreting antibodies recognizing huCD137 to enable CD137-CD137L interactions was determined by ELISA. These analyzes were performed initially in ELISA format. Human CD137-muIgG2bu was applied to the plates at a concentration of 0.2 µg/ml, 100 µl/well. Serial dilutions of monoclonal antibody 20H4.9-IgG1 or control antibodies, diluted in PBS-T and 1% bovine serum albumin, were added to the plates. CD137L-CD8 fusion protein was added to the chambers at a concentration of 0.2 µg/ml. Antibody binding was detected with a biotinylated anti-CD8 antibody (0.2 µg/ml, Ancell Corporation, Bayport, Minnesota). After several washes, streptavidin-alkaline phosphatase (1:2000) and pNPP were added to detect bound antibodies and the plates were read at 405 nm.

To confirm that the selected antibodies did not alter CD137-CD137L binding, additional characterization of the purified antibodies was performed using BIAcore analyses. All experiments were performed on a BIAcore 3000 device (BIAcore Inc., Piscataway, New Jersey). Human CD137 was covalently immobilized at high density on the surface of a carboxy-methylated dextran BIAcore sensor chip (BIAcore Inc., Piscataway, New Jersey). The injection was performed at a concentration of 2 µg/mL in 10 mM acetate buffer, pH 5.0. Unoccupied active esters were then blocked by injecting excess ethanolamine. Surface restoration was performed with 10 mM glycine, pH 2.0.

Purified anti-CD137 antibody samples were diluted to concentrations between 200 and 1000 nM using HEPES-buffered saline, pH 7.4, supplemented with 0.15 M NaCl and 0.005% surfactant P20 (HBS-EP). Human CD137L-CD8 fusion proteins (huCD137L) were used as a source of CD137 Uganda. Experiments were performed in which huCD137L was injected before anti-CD137 antibody, or vice versa. The injection was performed with a flow rate of 5 L/min. Bound ligand and antibodies were removed by resuspension with 10 mM glycine buffer, pH 2.0.

Purification of human T-cells: T-cells or PBMCs were obtained from healthy human donors. Blood was collected in EDTA, suspended in clarification buffer (RPMI containing 2.5 mM EDTA, 10 µg/ml polymyxin B), placed in lymphocyte separation medium (LSM, Mediatech Inc., Herndon, Virginia) and centrifuged at 1800 rpm for 25 minutes. Cell supernatants were harvested and centrifuged at 1500 rpm for 10 minutes. Then, cell pellets were resuspended in clarification buffer and washed with sheep red blood cells (SRBC, 1:10 dilution) and incubated on ice for 1 hour. Cells were then plated in LSM and centrifuged at 2500 rpm for 25 minutes. Cell contact surfaces were removed and SRBCs were lysed using SRBC lysis buffer. Isolated T-cells were washed and resuspended in 10% FBS/RPMI.

Flow cytometry analyses: Binding of anti-human CD137 antibodies to CD137 expressed on cells was determined by flow cytometry. Human T-cell leukemia cell line (CEM) or macaque monkey peripheral blood monocytic cells (PBMC) were used for these studies. These cells do not constitutively express CD137, but the receptor can be induced by stimulation with phorbol myristate (PMA, 10 ng/ml) and ionomycin (1 µM) for 18 h. Cells were then washed and incubated with various concentrations of antibody in staining buffer (phosphate buffered saline, PBS, plus 1% FCS and 0.01% sodium azide). Antibody binding to stimulated or unstimulated cells was detected using goat anti-human IgG conjugated to fluorescein (FITC) or phycoerythrin (PE) (Jackson Immunoresearch, West Grove, Pennsylvania). To confirm CD137 expression, a fusion protein consisting of the extracellular domain of CD 137 Uganda and mouse CD8 (Ancell Corporation, Bayport, Minnesota) was used, followed by incubation with PE-conjugated anti-mouse CD8 (BD Pharmingen, San Diego, California). Samples were then fixed in 1% formalin, kept at 4°C and then read by flow cytometry.

Functional assays: Originally human T-cells or monkey PBMCs obtained from healthy donors were stimulated with immobilized anti-CD3 antibody to provide the first signal for T-cell activation and then co-stimulated with human anti-human CD137 antibodies. As a non-specific control, humanized anti-carcinoma antibody (BR96) was used at the same antibody concentration. Plates were coated with anti-CD3 antibody (0.5-1 ug/ml) at 4°C overnight. The next day, T-cells or PBMCs were added to the plate at concentrations of 1-1.5 xl0<5>/well. IFN-γ synthesis was measured after 72 hours of culture at 37°C either by cytometric bead array (CBA) or by ELISA.

Cytokine analyses

ELISA: After T-cell stimulation at different times, the plates were centrifuged and the medium was removed. Cytokine levels were determined by ELISA according to the manufacturer's instructions (BD Pharmingen, San Diego, California). Briefly, test samples and controls were added to anti-cytokine-coated 96-well plates. After incubation for 2 hours at room temperature, the plates were washed 3 times in PBS-T and then incubated first with the working indicator antibody and then with the addition of medium. Absorbance was read at 405 nm and concentrations were calculated from the standard curve.

Cytometric bead array: The next procedure used to determine cytokinin production in vitro was flow cytometry using a cytometric bead array (CBA) developed at BD Pharmingen. Levels of IFN-7, IL-2, IL-5, IL-4, IL-10 and TNF were measured in culture supernatants according to the manufacturer's instructions. The results were analyzed by flow cytometry using CBA analysis software.

Results

Hybridomas secreting antibodies that showed binding to human CD137 were further propagated and subcloned. Secreted antibodies were purified and tested for their ability to bind to huCD137 and enable CD137-CD137L interaction. Of the anti-human CD137 antibodies evaluated, the monoclonal antibody 20H4.9-IgG1 was selected for further evaluation based on its binding profile and non-blocking properties. The 20H4.9-IgG1 antibody is IgGi kappa as determined by ELISA using alkaline phosphatase anti-human IgGi, 2, 3,4 and anti-kappa and lambda reagents (Southern Biotech, Birmingham, Alabama). FIG. 8 (FIG. 8A-binding to human CD137 by ELISA; FIG. 8B-effect of monoclonal antibody 20H4.9-IgGl on CD137-CD137L interaction) provides an initial characterization of monoclonal antibody 20H4.9-IgGl. The ability of serial dilutions of monoclonal antibody 20H4.9-IgG1, 26G6 (blocking anti-CD137 antibody), or tetanus toxoid (TT, negative control) to alter CD137 binding to CD137L was determined. Monoclonal antibody 20H4.9-IgGl in concentrations up to 10 µg/ml did not block CD137L binding, while monoclonal antibody 26G6 inhibited binding at concentrations >0.37 µg/ml.

Monoclonal antibody 20H4.9-IgG1 was also tested for reactivity against CD 137 expressed on human T-cells (CEMs) and in macaque monkey peripheral blood monocytic cells (PBMCs) stimulated with PMA and ionomycin. Previous studies have shown that CD137 is up-regulated on T-cells after activation with PMA and ionomycin. Control molecules consisted of an irrelevant human IgG antibody (negative control) or a CD137L-CD8 fusion protein (positive control, BD Pharmingen, San Diego, California). The results of these studies showed that monoclonal antibody 20H4.9-IgG1 binds to activated human CEMs and macaque monkey PBMCs, with minimal binding to unstimulated cells. Similar percentages of positive cells were determined with monoclonal antibody 20H4.9-IgGl and CD137L. FIG. 9 shows the results obtained by showing the binding of monoclonal antibody 20H4.9-IgG1 to PMA-ionomycin-stimulated human or macaque monkey cells. Human CEMs (FIG. 9A) or monkey PBMCs (FIG. 9B) were incubated with 20H4.9-IgG1 or human CD137L fusion protein. Secondary antibodies were added and samples were read by flow cytometry.

Next, it was determined whether monoclonal antibody 20H4.9-IgG1 could induce an increase in IFN-γ in costimulatory assays in the presence of anti-CD3 stimulation, which is a key functional effect desired for an agonistic CD137 antibody. The costimulatory activity of the monoclonal antibody 20H4.9-IgGl was determined in functional tests in human lymphocytes and monkey lymphocytes. Based on initial results, a concentration of 20 ug/ml anti-CD137 antibody (excess antibody) was used in these trials. Anti-CD3 antibody levels between 0.2-1 ug/ml were tested, resulting in 10-20% of CD137-positive lymphocytes. The levels of IFN-γ in the supernatants were measured after 72 hours of culture. As shown in FIG. 10, monoclonal antibody 20H4.9-IgG1 enhanced IFN-γ synthesis in both human and monkey costimulatory assays to levels significantly greater than controls. The results of tests performed with T-cells isolated from 8 healthy human donors showed that in six of them, the monoclonal antibody 20H4.9-IgG1 enhanced the synthesis of IFN-γ between 2.2-4.3-fold compared to controls. One of the other two donors showed a 1.6-fold increase. The level of increase was superior to that observed with hu39E3.G4, a humanized anti-CD137 antibody reported in published PCT application WO04/010947 (reference herein), which showed an increase in IFN-γ in 5 of a total of 8 donors and at levels lower than monoclonal antibody 20H4.9-IgGl (1.5-2-fold increase) (FIG. 10A). In costimulatory studies in monkeys, the monoclonal antibody 20H4.9-IgG1 also showed increased functional activity, resulting in a significant increase in IFN-γ compared to controls (FIG. 10B). As in the human studies, the increase in IFN-γ was greater than with hu39E3.G4.

Induction of TNF synthesis above control levels has also been reported in human cultures, although at much lower levels than IFN-γ. TNF levels induced by anti-

CD3 antibody (baseline) were about 20-50 times lower than baseline for IFN-γ. Monoclonal antibody 20H4.9-IgGl induced a ~2- to 4.7-fold increase in 3 out of a total of 8 donors. Again, hu39E3.G4 (tested in parallel) induced a ~2-fold increase in the same donors, but at lower levels. The other cytokines tested, IL-2, IL-5, IL-10 and IL-4 were not significantly changed with any of the treatments.

Together, these studies demonstrate that monoclonal antibody 20H4.9-IgG1 exhibits functional activity that is desirable in both humans and monkeys via induction of Th1-type responses. Notably, given that in vivo anti-tumor activity correlates with the ability of anti-CD137 antibodies to induce IFN-γ synthesis, these results support the choice of the 20H4.9-IgG1 monoclonal antibody for isotype switching.

Example 2: In vitro characterization of monoclonal antibody 20H4.9-IgG4

Based on its binding kinetics, inability to block the CD 13 7-CD137L interaction, and functional effects on human T-cells, monoclonal antibody 20H4.9-IgG1 was selected for conversion to the IgG4 form. The IgG4 form of the monoclonal antibody 20H4.9-IgG1 is 20H4.9-IgG4 (shown in FIG. 3 and 4).

The second phase of these studies included a comparison of the in vitro properties of the monoclonal antibody 20H4.9-IgG4 and the monoclonal antibody 20H4.9-IgG1. In this part, the binding kinetic properties and functional effects of both antibodies in human lymphocytes and monkey lymphocytes are described.

Binding kinetics

The kinetic properties of anti-human CD 137 antibodies were determined by surface plasmon resonance using a BIAcore 3000 device. The antigen, human CD137-mouse IgG2a, is covalently immobilized at low density on the surface of the CM5 sensor chip. Monoclonal antibody 20H4.9-IgG4 and monoclonal antibody 20H4.9-IgG1 were injected at concentrations between 25 and 200 nM. FIG. 11 shows injections of 100 nM monoclonal antibody 20H4.9-IgG1 and monoclonal antibody 20H4.9-IgG4. Results calculated using BIAevaluation software (bivalent model, analysis based on general curve fitting) gave kinetic parameters that were similar for both antibodies (see Table 1). The dissociation constants KD for monoclonal antibody 20H4.9-IgG1 and monoclonal antibody 20H4.9-IgG4 were determined to be 11.2 and 16.6 nM, respectively. Under similar experimental conditions, monoclonal antibody 20H4.9-IgG4 did not bind to murine 4-IBB.

Flow cytometry analyses

The binding of the biotinylated monoclonal antibody 20H4.9-IgG4 at concentrations ranging from 0.32 ng/ml to 5 ug/ml to CEM cells ± PMA-ionomycin was tested. Monoclonal antibody 20H4.9-IgG4 bound to PMA-ionomycin-stimulated CEM cells in a concentration-dependent manner. Saturation was achieved with 0.2 ug/ml. On the other hand, as shown for its parent molecule, monoclonal antibody 20H4.9-IgG1, monoclonal antibody 20H4.9-IgG4 did not bind to CEM cells not stimulated with PMA-ionomycin (FIG. 12). Concentration-dependent binding of monoclonal antibody 20H4.9-IgG4 was demonstrated in PMA-ionomycin-stimulated CEM cells (FIG. 2). The samples were read by flow cytometry.

Cell/functional assays

To confirm that the isotype switching process of monoclonal antibody 20H4.9-IgG1 did not alter antibody activity, in vitro assays were performed to compare the activity of monoclonal antibody 20H4.9-IgG4 with the parent monoclonal antibody 20H4.9-IgG1 in monkey PBMC and human T-cells. The functional effects of monoclonal antibody 20H4.9-IgG4 on human T-cells or monkey PBMCs were determined and compared with the parent molecule, monoclonal antibody 20H4.9-IgG1. Primary human T-cells or monkey PBMCs obtained from healthy donors were stimulated with anti-CD3 antibody (0.5 ug/ml-1 ug/ml) +/- anti-human CD137 antibodies. IFN-γ synthesis was measured after 72 hours in culture at 37°C by cytometric bead array (CBA) for human samples or by ELISA for monkey samples. Antibodies were tested in costimulatory assays in the presence of suboptimal concentrations of anti-CD3 antibody (1 µg/ml) or cocavalin A (1 µg/ml) (donors only M5170 and 81). The results are expressed as the number of times the values increased compared to the controls in pg/ml. Due to variable baseline response among donors, results were normalized to control treatments (=1). FIG. 13A shows the results for human T-cells and FIG. 13B shows the results for monkey PBMCs. As shown in FIG. 13A-13B, monoclonal antibody 20H4.9-IgG4 showed costimulatory properties by producing higher levels of IFN-γ in human and monkey cells compared to controls. The level of increase in IFN-γ synthesis was similar to that of its parent molecule in human and monkey samples.

Next, the effect of antibody cross-linking on the functional effects of the monoclonal antibody 20H4.9-TgG4 was determined. It has been shown that cross-linking of antibodies can result in enhancing their ability to transmit signals. Therefore, an assay was performed to determine the functional activity of several groups of monoclonal antibody 20H4.9-IgG4 ± anti-human IgG antibody. As shown in FIG. 14A, a significant increase in IFN-γ synthesis was noted for all tested groups in the absence of cross-linking antibodies, with a plateau at a concentration of 400 ng/ml. The increase in IFN-γ synthesis under the influence of the em monoclonal antibody 20H4.9-IgG4 was further enhanced by the addition of an anti-human IgG cross-linking antibody as shown in FIG. 14B. Different groups of monoclonal antibody 20H4.9-IgG4 had similar cellular activities.

Thus, cross-linking of the monoclonal antibody 20H4.9-IgG4 resulted in an increase in the ability of the antibody to induce IFN-γ synthesis. Cross-linking of antibodies in vivo can take place on cellular receptors for the Fc part of immunoglobulins or via antibody dimerization. The monoclonal antibody 20H4.9-IgG4 is of the IgG4 isotype, which, compared to other IgG isotypes, has a low affinity for Fc receptors. However, IgG4 can bind to FcγRI (CD64) which is expressed on monocytes and neutrophils.

Two other approaches were used to further characterize the monoclonal antibody 20H4.9-IgG4: (i) effect on T-cell survival and (ii) effect on cyclin D2 expression. To determine whether monoclonal antibody 20H4.9-IgG4 can induce signaling through CD 137 on human T-cells and provide costimulatory T-cell signals leading to cell survival and expansion, human T-cells stimulated with anti-CD3 antibodies +/− monoclonal antibody 20H4.9-IgG4 at concentrations known to induce synthesis IFN-7 was stained with annexin-V and propidium iodide to determine the number of viable cells (annexin V/propidium iodide negative), as well as cyclin D2 to determine its effect on cell cycle progression. FIG. 15 shows the mean of the results of 4 different groups of monoclonal antibody 20H4.9-IgG4 on cyclin D2 expression and T-cell survival. Concentrations of monoclonal antibody 20H4.9-IgG4 of 0.4-10 ug/ml resulted in an approximately 1.8-2-fold increase in the number of viable cells and produced a significant increase in the number of cyclin D2-expressing T cells (2.5-3-fold).

Example 3: In vivo evaluation of 4-IBB antibodies in a pharmacodynamic model in macaque monkeys

This example illustrates the ability of monoclonal antibody 20H4.9-IgG4 and monoclonal antibody hu39E3.G4 to enhance the antigen-specific immune response induced by DNA vaccines.

Material and methods

Experimental Animal Groups: Female and male macaque monkeys (2.5 to 5.0 kg) were obtained from Charles River BRF (Houston, Texas) for this study and were assigned in pairs. Each experimental group consisted of 4 males and 2 females who were randomly divided into groups according to body weight. The experimental groups were as follows: Group 1 - SIV gag and PSA DNA vaccine (2 mg each), day 0, 28, 56, i.m., plus saline as control, i.v., on days 12, 15, and 19;

Group 2 - SIV gag and PSA DNA vaccine (2 mg each), day 0, 28, 56, i.m., plus monoclonal antibody hu39E3.G4, i.v., on days 12, 15, and 19;

Group 3 - SIV gag and PSA DNA vaccine (2 mg each), day 0, 28, 56, i.m., plus monoclonal antibody 20H4.9-IgG4, i.v., on days 12, 15, and 19;

Group 4 - untreated control group.

Immunizations and Antibody Treatments: PSA and SIV gag DNA vaccines were obtained from David B. Weiner, Department of Pathology and Laboratory of Medicine, University of Pennsylvania. (See, Kim et al., Oncogene 20, 4497-4506 (2001); Muthumani et al., Vaccine 21, 629-637 (2003).)

Monkeys were immunized intramuscularly with PSA and SIV gag DNA constructs (2 mg/construct/immunization) simultaneously, followed by two immunizations 4 weeks apart (days 0, 28, and 56). Twenty days after the initial immunization, treatment with monoclonal antibody 20H4.9-IgG4 or monoclonal antibody hu39E3.G4 was started. Antibodies were administered i.v., 50 mg/kg, on days 12, 15 and 19 after the first immunization. This schedule was chosen because it has been shown to suppress the antibody response to monoclonal antibody hu39E3.G4.

Clinical pathology

During the test, physical examinations were performed on all monkeys by competent veterinarians. Blood samples for hematological and serum chemistry analyzes were collected before vaccinations and then 12, 42, 70, 97, 134 and 168 days after immunizations.

Immunological analyses

To determine the effect of these therapeutic regimens on immune responses, an ELISPOT assay was used to determine IFN-γ production by antigen-specific stimulated lymphocytes. Blood samples for ELISPOT analyzes were collected before vaccinations and then 12, 42, 70, 97, 134 and 168 days after immunizations. Synthetic peptides corresponding to the entire sequences of SIV gag and PSA antigens were used for ex vivo stimulation of PBMCs.

Results

Antigen-specific cells secreting IFN-γ in response to PSA or SIV gag peptides were quantified by ELISPOT. FIG. 16 (FIGS. 16A-16D) illustrate the results obtained from Groups 1-4, respectively. The level of PSA response was very low in all groups, indicating that the vaccine itself did not induce a measurable and sustained immune response compared to non-vaccinated animals. On the other hand, SIV gag vaccination alone resulted in significant numbers of antigen-specific IFN-γ-secreting cells that increased over time (FIG. 16A). Untreated animals (non-vaccinated) showed 100-1,000 dots/10<6>PBMC throughout the study (FIG. 16D). These results established a response threshold to the vaccine; animals showing <1,000 dots/10<6>PBMC were determined to have no response. In the group of animals that received the vaccine, 5 of 6 monkeys showed an increased response over time, with a mean number of spots after the third immunization (day 70) of 1,727 spots/10<5>PBMC (SD-242, range=1,403-1,968 spots/10<6>PBMC). One monkey was determined as a non-responder (620 dots/million PBMC). Since MHC typing was not performed in these studies, it is likely that the lack of T cell response to the vaccine in some monkeys may be due to MHC mismatches. Remarkably, at day 70, 4 out of 6 animals treated with SIV gag-plus monoclonal antibody 20H4.9-IgG4 had significantly higher numbers of IFN-γ puncta (FIG. 16C) compared to control animals (FIG. 16D) and to macaque monkeys immunized with DNA vaccine alone (FIG. 16A). The mean number of spots after the third immunization for the group treated with monoclonal antibody 20H4.9-IgG4 was 3,465 spots/10<6>PBMC (SD=1,236, range=2,070-4,780 spots/10<6>PBMC). Two monkeys in that group did not respond to the vaccine (< 800 spots/million PBMC). After the third immunization (day 70), treatment with monoclonal antibody hu39E3.G4 plus DNA vaccine resulted in a response in 6 of a total of 6 animals with a mean number of dots/10<6>PBMC of 2,348 (SD=588, range=1,738-3,283) (FIG. 16B). For this group, the range of spot counts was lower compared to macaque monkeys treated with monoclonal antibody 20H4.9-IgG4.

Treatment with monoclonal antibody 20H4.9-IgG4 and monoclonal antibody hu9E3.G4 was well tolerated and did not result in any significant change in clinical symptoms, clinical herniation, or hematological parameters compared to control monkeys.

These results show that treatment with the monoclonal antibody 20H4.9-IgG4 in combination with the DNA vaccine caused an in vivo increase in the magnitude of the specific cellular response to the test antigen compared to controls or to treatment with the monoclonal antibody hu39E3.G4, as measured by antigen-specific IFN-γ-secreting cells. Given that only one antibody dose level and one dosing regimen were used in the preliminary trials, maximal responses were likely not induced and further work is needed to optimize the conditions. It is clear, however, that even with this unoptimized protocol, an increase in the cellular response to test antigens was achieved using the monoclonal antibody 20H4.9-IgG4, indicating that modulation of CD137 function may be a suitable approach to increase the efficacy of DNA vaccines.

Although the invention has been described in some detail by way of illustration and example for the sake of clarity and understanding, it will be understood that certain changes and modifications may be made within the scope of the appended claims.

Claims (9)

1. A fully human monoclonal antibody or antigen-binding portion thereof, which specifically binds to human 4-IBB and which contains a light chain variable region and a heavy chain variable region, characterized in that: said light chain variable region comprises CDR1 comprising amino acid residues 44-54 of SEQ ID NO:6, CDR2 comprising amino acid residues 70-76 of SEQ ID NO: 6 and CDR3 comprising amino acid residues 109-119 of SEQ ID NO:6; and said heavy chain variable region comprises CDR1 comprising amino acid residues 50-54 of SEQ ID NO:3, CDR2 comprising amino acid residues 69-84 of SEQ ID NO:3 and CDR3 comprising amino acid residues 117-129 of SEQ ID NO:3. 2. A fully human monoclonal antibody or antigen-binding part thereof according to claim 1, characterized in that: said light chain contains a variable region containing amino acid residues 21-129 of the sequence SEQ ID NO:6; and said heavy chain comprises a variable region comprising amino acid residues 20-140 of SEQ ID NO:3. 3. A fully human monoclonal antibody containing a light chain and a heavy chain according to claims 1 and 2, characterized in that said light chain contains amino acid residues 21-236 of sequence SEQ ID NO: 6 and said heavy chain contains amino acid residues 20-467 of sequence SEQ ID NO: 3. 4. A pharmaceutical composition containing: a fully human monoclonal antibody or an antigen-binding part thereof according to claim 1; and a pharmaceutically acceptable carrier. 5. A pharmaceutical composition containing: a fully human monoclonal antibody according to claim 3; and a pharmaceutically acceptable carrier. 6. Use of a therapeutically effective amount of a fully human monoclonal antibody or antigen-binding portion thereof according to claim 1 for the manufacture of a medicament for the treatment of cancer in a subject. 7. An isolated polynucleotide containing a nucleotide sequence encoding the amino acid sequence of amino acid residues 20-467 of sequence SEQ ID NO: 3 and the amino acid sequence of amino acid residues 21-236 of sequence SEQ ID NO:6. 8. Polynucleotide according to patent claim 7, which contains the nucleotide sequence SEQ ID NO: 1. 9. Polynucleotide according to patent claim 7 containing the nucleotide sequence SEQ ID NO: 4.